Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess14293 J. Oleo Sci. 64, (6) 603-616 (2015)
Variation of Lipids and Fatty Acids of the Japanese Freshwater Eel, Anguilla japonica, during Spawning Migration Hiroaki Saito1＊ , Hiroaki Kurogi2, Seinen Chow3 and Noritaka Mochioka4 1
Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, 921-8836, Japan. Yokosuka-Chosha, National Research Institute of Aquaculture, 6-3-1 Nagai, Yokosuka 238-0316, Japan. 3 National Research Institute of Fisheries Science, 2-12-4, Fukuura, Kanazawa-Ku, Yokohama 236-8648, Japan. 4 Kyushu University, 6-10-1, Hakozaki, Fukuoka 812-8581, Japan. 2
Abstract: The lipid and fatty acid composition of the muscle of the wild Japanese freshwater eel, Anguilla japonica, was analyzed between the initial and terminal stages of spawning migration to clarify the relationship between lipid physiology and maturation. Triacylglycerols were the only major component in the initial-phase eels, which contained high levels of lipids, while comparatively low triacylglycerol levels were observed in terminal-phase eels (Mariana silvers) at spawning area. Significant levels of plasmalogens were found in its phosphatidylethanolamine, different from other common fish species, which have their little levels. The major fatty acids in A. japonica depot triacylglycerols were 14:0, 16:0, 18:0, 16:1n-7, 18:1n7, 18:1n-9, and 18:2n-6. Noticeable levels of 20:4n-6, EPA, 22:5n-3, and DHA were also found in initialphase sample TAG at the yellow and initial silver stages. High 18:2n-6 levels in all A. japonica lipids were similar to those in other common freshwater fishes. In all A. japonica tissue phospholipids, high levels of n-6 and n-3 PUFA, such as 20:4n-6, EPA, 22:5n-3, and DHA, were observed except for the matured terminal female sample. High n-6 PUFA levels in terminal-phase samples caught at the spawning area suggest that A. japonica maintains and uses initial fatty acids from inland waters without feeding during long spawning migrations. The post-spawning sample, containing low levels of 20:4n-6 and DHA with unusually high levels of its degradation products (18:3n-6, 20:2n-6, and 18:4n-3), indicates that A. japonica may finally use its most important PUFA as energy for spawning before ending its life. Key words: arachidonic acid, chemoecology, spawning migration, fatty acid, marine lipid, monounsaturated fatty acid, polyunsaturated fatty acid 1 INTRODUCTION Low levels of n-6 PUFA are generally found in marine fish lipids. For example, 18:2n-6 and 20:4n-6 are minor components in their lipids1−5）, except for some marine herbivorous animals that prefer and consume seaweed and specifically accumulate 20:4n-6 in their tissue lipids6, 7）. In contrast, freshwater fishes consume terrestrial prey. Therefore, their lipids specifically contain high levels of n-6 PUFA. The n-6 PUFA are always observed as major PUFA in terrestrial animal lipids. For example, 18:2n-6 and 20:4n-6 are characteristically contained in freshwater fishes. In particular, 18:2n-6 is observed in their depot neutral lipids and 20:4n-6 is mainly found in the tissue phospholipids of fish such as common carp, Cyprinus
carpio8, 9）, Carassius auratus auratus9）; rainbow trout, Salmo gairdnerii8）; eel, Anguilla japonica8）; and Indian featherback fish, Notopterus notopterus10）. This suggests that 18:2n-6 and 20:4n-6 are useful as lipid biomarkers for terrestrial8−10）or herbivorous animals6, 7）. Wild eels（Anguilla spp., Anguillidae）mainly dwell in Abbreviations: ARA, arachidonic acid; DMOX, 4,4-dimethyloxazoline; DHA, docosahexaenoic acid; EPA (IPA); icosapentaenoic acid; GC/MS, gas chromatography/mass spectroscopy; MUFA, monounsaturated fatty acids; NMID, non-methylene-interrupted dienes; NMR, nuclear magnetic resonance; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PUFA, polyunsaturated fatty acids; TAG, triacylglycerols; TFA, total fatty acids; TL, total lipids.
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Correspondence to: Hiroaki Saito, Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, 921-8836, Japan. E-mail: [email protected] Accepted February 2, 2015 (received for review December 26, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 603
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freshwater areas, take prey in lakes and rivers, and their fatty acids contain significant levels of 18:2n-6 and 20:4n68, 11）. Freshwater eels of the genus Anguilla metamorphose into the silver stage from yellow stage upon onset of maturation, cease feeding and start reproductive migration. European eels（Anguilla anguilla）undertake a 5000-km spawning migration from Europe to the Sargasso Sea12, 13）, and details of their migration have been gradually revealed. Laboratory experiments support the fact that European eels are capable of swimming a distance of over 5500 km without resting and feeding14） and the starving silver eels are able to reach their spawning grounds by using their stores of internal fat15）. In the Pacific Ocean, the Japanese eel （Anguilla japonica Temminck & Schlegel, Anguillidae） may also depend on its store of fat for spawning migrations without feeding16） because the alimentary tracts of silver eels are degenerated17, 18）. Recently, adult matured Japanese eels（terminal phase）have been caught in the West Mariana Ridge, which is its spawning area16）. An interesting comparison of the fatty acids of spawning-area eels（terminal phase） can be made with those of Japanese eels at the , which are collected yellow and silver stages （initial phase） in rivers and lakes and presumably influenced by terrestrial . prey lipids（Lake Ogawara19）, and Amori River20）） The present manuscript deals with the detailed lipid composition and analysis of the fatty acid components of the major lipid classes of A. japonica between these ‘Mariana silvers’ （terminal phase） and Japanese eels at the yellow and initial silver stages（initial phase）in order to clarify its lipid physiology and to find out its feeding behavior during reproductive migrations.
2 MATERIALS AND METHODS 2.1 Materials Specimens of yellow and initial silver stages A. japonica （yellow-stage eels: samples No. 1–4, silver-stage eels: samples No. 5–10 in Table 1）were caught in three Japanese rivers（the Koyama River, the Yakkan River, and the Akigawa River）and Okinoshima Island near Japanese coasts of the Japan Sea and the Beppu Bay in Seto-Inland Sea in the northern Pacific Ocean. Specimens of completely matured Mariana silver eel A. japonica（terminal-phase stage eels: samples No. 11–17 in Table 1）were caught by the research vessels‘Kaiyo-maru’and“Hokko-maru”, which belongs to the Fisheries Agency, Tokyo, Japan, in the West Mariana Ridge in the mesopelagic zone of the northwestern Pacific Ocean. Research cruises were performed during 2008 and 2009, and adult Japanese eels were captured by a large mid-water trawl net in the southern part of the West Mariana Ridge（ Table 1）16）. The samples of A. japonica listed in Table 1 were frozen at −40℃ until extraction. After measurements of body
length and weight of A. japonica samples, the following tissues and organs, such as muscle and liver, were dissected from each fish. 2.2 Lipid extraction and analysis of lipid classes Each individual（muscle）was immersed in a reagent mixture containing chloroform and methanol（2:1, v/v）, and homogenized in the same solvents, and each lipid of a homogenized sample was then extracted according to the Folch procedure21）. Each crude lipid was separated into classes on a silicic acid column（Merck and Co. Ltd., Kieselgel 60, 70 - 230 mesh）, and the constituent lipids were quantitatively analyzed by gravimetric analysis of the column chromatography fractions（Table 2）22）. The lipid classes from each lipid fraction were identified by comparison of the retention factor（Rf）values of standards using plate thin-layer chromatography （Merck & Co. Ltd., Kieselgel 60, thickness of 0.25 mm for analysis） , and by the characteristic peaks observed in nuclear magnetic resonance 22） . All sample lipids were dried under argon at room （NMR） temperature, and stored at −40℃ under an argon atmosphere. 2.3 NMR spectrometry and the determination of lipid classes Spectra were recorded on a GSX-270 NMR spectrometer （JEOL Co. Ltd., Tokyo, Japan）in a pulsed Fourier transform mode at 270 MHz in a deuterochloroform solution using tetramethylsilane as the internal standard22）. 2.4 Preparation of methyl esters and analysis by gasliquid chromatography Fatty acids present in TAG, PE, and PC fractions were converted to fatty acid methyl esters by direct transesterification with methanol containing 1％ concentrated hydrochloric acid under reflux for 1.5 hr, as previously reported22）. These methyl esters were purified using silica gel column chromatography by elution with dichloromethane/ n-hexane（ 2/1, v/v）. The composition of the fatty acid methyl esters were determined by gas liquid chromatography using an HP 6890N gas chromatograph（Agilent Technology Co., Yokogawa Electric Corporation, Tokyo, Japan） equipped with an omegawax-250 fused silica capillary column（30 m×0.25 mm i. d. ; 0.25 μm film, Supelco Japan Co. Ltd, Tokyo, Japan）. The temperatures of the injector and the column were held at 250 and 210℃, respectively, and the split ratio was 1:76. Helium was used as the carrier gas at a constant inlet rate of 0.7 mL/min. Quantitation of individual components was performed by means of an HP ChemStation System （Rev. B.03.02 ［341］version, Yokogawa HP Co. Ltd.） electronic integrator（Tables 3–6）.
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Table 1 Locality of capture and biological data of three types of wild Japanese eel, Anguilla japonicus.
Variation of Lipids and Fatty Acids of the Japanese Freshwater Eel, Anguilla japonica, during Spawning Migration
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Table 2 The lipid contents and lipid classes of the muscles of three types of wild Japanese eel, Anguilla japonica.
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Table 3 The fatty acid composition of muscles of the common yellow Eels (%)a.
Variation of Lipids and Fatty Acids of the Japanese Freshwater Eel, Anguilla japonica, during Spawning Migration
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Table 4 The fatty acid composition of muscles of the silver Eels (%)a.
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Table 5 The fatty acid composition of muscles of the mature 2008 eel (%)a.
Variation of Lipids and Fatty Acids of the Japanese Freshwater Eel, Anguilla japonica, during Spawning Migration
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Table 6 The fatty acid composition of muscles of the mature 2009 eel (%)a.
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Variation of Lipids and Fatty Acids of the Japanese Freshwater Eel, Anguilla japonica, during Spawning Migration
2.5 Preparation of 4,4-dimethyloxazoline derivatives （DMOX）and analysis of DMOX by gas chromatography - mass spectrometry （GC-MS） DMOX derivatives were prepared by adding an excess amount of 2-amino-2-methylpropanol to a small amount of the fatty acid methyl esters in a test tube under an argon atmosphere. The mixture was heated at 180℃ for 18h22, 23）. Analysis of the DMOX derivatives was performed on an HP G1800C GCD Series II GC-MS（Hewlett Packard Co. Ltd., Yokogawa Electric Corporation, Tokyo, Japan）and an Agilent 6890N-5973N GC-MS System（Agilent Technologies Inc., Tokyo, Japan）equipped with the same capillary column for determining the fatty acids with an HP WS（HP Kayak XA, G1701BA version, PC workstations and MSD ChemStation D.03.00.611） . The temperatures of the injector and the column were held at 230 and 210℃, respectively. The split ratio was 1:76 and the ionization voltage was 70 eV. Helium was used as the carrier gas at a constant inlet rate of 0.7 mL/min. Fatty acid methyl esters were identified by comparing the mass spectral data of the methyl esters and DMOX derivatives obtained by GC-MS. 2.6 Statistical analyses The determination of fatty acids by gas-liquid chromatography was based on more than two replicates（n＝2–12 in Tables 3–6）. Lipid content, lipid class, and fatty acid levels（％ of wet weight, ％ of total lipids, and ％ of total fatty acids） are expressed as mean values±standard errors. The mean total PUFA, total n-4 PUFA, total n-6 PUFA, total n-3 PUFA levels of the samples were compared with each other. Significant mean differences were determined using a one-way analysis of variance（ANOVA）. Tukey’ s multiple procedure was used to compare the differences among mean values. Differences were regarded as significance level of p＜0.05.
3 RESULTS 3.1 Lipid content in the three different types of A. japonica The three types of A. japonica samples（yellow stage samples 1–4: silver stage samples 5–10: and Mariana silver samples 11–17）were caught in the Japan Sea and the northwestern Pacific Ocean, as shown in Table 1. Except for sample 4, the muscle lipid content in two types of samples caught in rivers and near Japanese coasts was significantly high（ samples 1–3: 12.8–16.3％ and samples 5–10: 14.4–20.6％）, while that in matured Mariana silver samples was comparatively low and fluctuated（ 2008 samples 11–14: 0.3–2.3％ and 2009 samples 15–17: 3.2– 9.7％ in Table 2）.
lipids（steryl esters, wax esters, diacylglyceryl ethers, TAG, sterols, and free fatty acids）and phospholipids（PE and PC）, as shown in Table 2. In all muscles of A. japonica caught near Japan（initial phase）, TAG（69.7– 94.0％ for samples 1–4 and 93.2–95.5％ for samples 5–10）was the only major component with trace or small levels of phospholipids. Medium levels of free fatty acids were found in two terminal-phase samples（13.4％ for sample 11 and 18.3％ for sample 13. Unusually medium levels of dimethyl acetals （DMA） in all muscle PE（13.4–22.0％ for samples 1–4, 9.5– 17.4％ for samples 5–10, and 3.1–15.2％ for samples 11–17） were observed（Tables 3–6） . 3.3 Fatty acid composition of A. japonica major TAG The mean values of the fatty acid compositions of the major purified lipids extracted from A. japonica are presented in Tables 3–7. At least fifty peaks were detected and identified, of which more than thirty consisted of more than 0.1％ total fatty acids（TFA）. Even-numbered carbon atoms, ranging from C14 to C22, were more abundant than odd-numbered and branched ones, which is similar to those of all other fish species. The kinds of fatty acids of all phases of A. japonica TAG were similar to each other. In the final-phase samples caught near the West Mariana Ridge, seven major components（more than about 3％ of TFA）were found in the final-phase A. japonica depot TAG, 14:0, 16:0, 18:0, 16:1n-7, 18:1n-7, 18:1n-9, and 18:2n-6, which are mostly saturated and monounsaturated fatty acids. Although the TAG contained various kinds of fatty acids, these seven components were detected at a level of about 3％ or more of TFA of the final-phase specimens. Noticeable levels of n-6 and n-3 long-chain PUFA such as 20:4n-6, EPA, 22:5n-3, and DHA were also found in the TAG of some final-phase samples. In contrast, several major PUFA were found in the TAG of the fatty initialphase samples, which were caught in the rivers and near Japanese coasts: EPA and 22:5n-3 with noticeable levels of some other PUFA such as 18:2n-6, 20:4n-6, 18:3n-3, and DHA. The levels of these PUFA were sometimes over about 3％ of TFA, in particular, in the case of EPA and 22:5n-3 in these samples（samples 1–3, 5, and 10） . 3.4 Fatty acid composition of A. japonica tissue phospholipids In the phospholipids of all samples, nine major components were found uniformly, 16:0, 18:0, 16:1n-7, 18:1n-7, 18:1n-9, 20:4n-6, EPA, 22:5n-3, and DHA, with noticeable levels of 14:0, 18:2n-6, and 18:3n-6, except for the terminal sample, No. 13, whose major fatty acids were expanded to various short-chain fatty acids, 14:0, 16:0, 18:0, 18:1n-9, 18:2n-6, 18:3n-6, 20:2n-6, 20:4n-6, 18:4n-3, and DHA.
3.2 Lipid Classes of A. japonica The TL were fractionated into various classes as neutral 611
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Table 7 Actual DHA levels in 100 g of wet tissue.
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4 DISCUSSION 4.1 Lipid content in three different types of A. japonica A. japonica caught near Japan （initial phase） accumulated high levels of lipids in the muscles: this characteristic feature is also generally observed in the other Anguilla species10, 19）. However, after long-distance spawning migrations, the Mariana silver eels（terminal phase）have lower lipid levels（ Table 2）. For example, samples 11 and 12 （male, 2008） and samples 13 and 14 （female, 2008） , having the lowest lipid levels completely lost their lipids, while samples 15 and 16（male, 2009）and sample 17（female, 2009）, which have several percentages of lipid content, may be able to maintain their lives. This difference in the Mariana silvers between 2008 and 2009 indicates that A. japonica may partly use its internal fat stores for long-distance migration over 2000–3000 km12, 14, 16） and may retain its depot lipids for multiple spawnings 24）. The 2008 samples, which completely lost their lipids（0.3–2.3％ for 11–14）may be exhausted by migration and spawning, while the 2009 samples（ 15–17）may be able to spawn several times and be close to ending its life. 4.2 Lipid Classes of A. japonica The occurrence of high levels of muscle TAG was due to the high levels of muscle lipid content because phospholipid levels in animal cells remain constant and the relative levels of depot lipids in fatty fishes are generally higher than those in lean animals4−7, 25, 26）. Medium levels of free fatty acids were specifically found in two terminal-phase, which probably degenerated from TAG to decomposed free fatty acids. The enzymatic degradation of A. japonica TAG may have actively proceeded at the terminal phase because a terminal-phase eel, which is probably unable to feed17, 18）, should obtain energy for spawning from its internal tissue lipids. Different from other fish species27, 28）, unusually medium levels of dimethyl acetals（ DMA）in muscle PE suggest that plasmalogen-type PE are biosynthesized in eel muscles similar to those in invertebrates such as mollusks25, 26）. A. japonica mainly biosynthesizes DMA 16:0, 18:0, and 18:1 for alkenyl ethers in PE plasmalogens, which is also similar to those of other common invertebrates25−27）. This is the characteristic profile of the freshwater Japanese eel. 4.3 Fatty acid composition of A. japonica major TAG High 18:2n-6 levels in all A. japonica TAG lipids were similar to those in other common freshwater fishes8−10）. The characteristically high levels of 18:2n-6 in their TAG are presumably influenced by those of the prey lipids in inland waters. Similar to the lipids of freshwater eel, carp8, 9）, and featherback 10）, which are typical freshwater fish species in rivers and lakes, all A. japonica had high levels of 18:2n-6. For example, its levels in the TAG of A. japonica of both phases were 1.5–5.3％ for samples 1–4, 1.0– 6.6％ for samples 5–10, 2.7–3.9％ for samples 11–14, and
2.0–3.1％ for samples 15–17. In particular, samples 11–17 showed comparatively high 18:2n-6 levels in the TAG even though they have inhabited marine environments for a long time1−4）. Simultaneously, the terminal-phase samples have markedly low levels of long-chain n-3 PUFA（EPA: 0.1– 1.2％ for samples 11–14 and 0.2–1.3％ for samples 15–17; DHA: 0.4–1.4％ for samples 11–14 and 0.7–2.0％ for samples 15–17）, different from those in other common marine fish species, which feed on marine prey, assimilate their lipids, and have high levels of these n-3 PUFA1−5, 29）. For example, high levels of DHA are usually observed in typical marine fishes, such as tuna, which are highly migratory fishes1, 2, 4）. These findings imply that the terminalphase samples are probably not dependent on marine prey lipids and are continuously influenced by only inland animal lipids15）. 4.4 Fatty acid composition of A. japonica tissue phospholipids Although the lipid contents between initial-phase samples（1–10）and terminal-phase samples（11–17）were different from each other, various n-6 PUFA（18:2 n-6, 18:3 n-6, 20:2 n-6, 20:3 n-6, and 20:4 n-6）and high levels of long-chain n-3 PUFA（EPA, 22:5 n-3 and DHA）in all sample PE were observed in both phases of the samples except for the terminal sample, No. 13. Similarly, the same trends were also found in PC, except for the terminal sample, No. 13. In particular, consistently high levels of 20:4n-6 and DHA in all sample PE and PC were observed in both phases of the samples. The 20:4n-6 levels in the phospholipids of A. japonica samples ranged as follows: 4.6–14.2％ for PE and PC of samples 1–4, 4.6–12.3％ for those of samples 5–10, 7.2–16.8％ for those of samples 11, 12, and 14, and 5.1–11.3％ for those of samples 15–17. The same trends are found for DHA: 8.0–19.1％ for PE and PC of samples 1–4, 9.5–20.4 for those of samples 5–10, 9.4–22.8％ for those of samples 11, 12, and 14, and 15.4–28.7％ for those of samples 15–17 except for the terminal sample, No. 13, whose n-3 and n-6 LC PUFA levels were the lowest. These phenomena suggest the importance and essentiality of 20:4n-6 and DHA for A. japonica 29−32） although even 20:4n-6 and DHA were consumed in the case of sample No. 13, which was presumably in its last mortal stage. As for LC n-3 PUFA, the same trends were observed in the marine eels, which have high levels of both EPA and DHA29）. 4.5 High levels of n-6 PUFA and degradation of 20:4n-6 and DHA in A. japonica tissue phospholipids Similar to other freshwater fishes9, 10）, the significant level of 20:4n-6 of all A. japonica polar lipids differed from that of common marine fish species1−5）. Different from the fatty acid compositions of marine origin larvae of tropical eels29）, high 20:4n-6 levels in the major polar lipids indicate 613
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that A. japonica is not dependent on marine prey animal lipids, which contain low levels of n-6 PUFA and high levels of n-3 PUFA1−5） except for the terminal sample, No. 13. With significant 18:2n-6 levels in TAG, the high levels of 20:4n-6 in polar lipids in terminal-phase A. japonica also indicate that A. japonica shows profiles of typical freshwater animal lipids even in the sea. This finding supports the assumption that A. japonica, which is carnivorous, migrates to spawning areas without feeding16） because high n-6 PUFA levels are only found in some marine herbivorous fishes6, 7）. In general, limited long-chain n-3 PUFA, such as EPA and DHA, are mainly found in marine fishes in higher trophic levels. In particular, DHA is the only major PUFA in highly migratory fishes1, 2, 4）. This means that both the EPA and DHA are terminal PUFA and many marine fishes only accumulate them1−5）. Interestingly, unusually high levels of 22:5n-3 were found in the polar lipids of all A. japonica. This is the specific lipid profile for A. japonica because only some mollusks, such as abalone and snails25, 34, 35）, show noticeable levels of 22:5n-3. High levels of 22:5n-3 in A. japonica lipids indicate its biosynthetic weakness of DHA, similar to those in the mollusks25, 34, 35） and other freshwater fishes8, 33）. In the spawning migration, the actual DHA levels in the tissue of A. japonica were markedly reduced in terminalphase eels （2008 Mariana silver eels 11–14） , which had finished migration and spawning, while those of A. japonica after migration（ 2009 Mariana silver eels 15–17）were similar to those in initial-phase eels. For example, the actual DHA amounts in the depot TAG were 0.4–1.4 mg （lipid content×TAG ratio×DHA ratio/100 g of wet tissue） in the final phase（the 2008 eels 11–14）while those were 34.6–369.7 mg and 160.8–644.9 mg in the initial phase （yellow eels 1–4 and silver eels 5–10） . In the case of 2009 samples （15–17） , the actual DHA amounts were 15.9–171.2 mg, similar to those in the two initial phase samples （Table 7） . Similar trends were observed in the phospholipids. For example, the actual DHA amounts in the tissue PE （lipid content×PE ratio×DHA ratio/100 g of wet tissue）at the final phase samples （the 2008 eels 11–14） were 3.6–5.8 mg, while those at the two initial phases（ samples 1–4 and 5–10）were 10.9–46.1, 3.8–17.8, and at the final phase samples（the 2009 eels 15–17）were 14.0–43.7 mg（Table 7）. This implies that A. japonica probably retain DHA during spawning migration by using other fatty acids such as saturated fatty acids and MUFA as energy sources. Eventually, however, it finally uses even the most important PUFA, DHA36−38）, for multiple spawnings, and then ends its life.
ACKNOWLEDGMENTS We are especially indebted to the captains and crew of the Research Vessels“Kaiyo-maru”and“Hokko-maru” of the Fisheries Agency for their cooperation and enthusiasm throughout the work. The authors also thank Ms. Sumiko Terada, Ms. Noriko Tsutsui, and Mr. Akihito Takashima for their skilled technical assistance. This work was supported in part by the research projects from the Fisheries Research Agency of Japan. H. S. performed all aspects of the study including the design of the experiments, analyzing the data, and writing the manuscript. H. K., S. C., and N. M. caught the animals and determined biological data.
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Chem. Soc. 77, 825-830 （2000） . 10）Mukhopadhyay, T.; Nabdi, S.; Ghosh, S. Lipid profile and fatty acid composition in eggs of Indian featherback fish Pholui（Notopterus notopterus Pallas）in comparison with body-tissue lipid. J. Oleo Sci. 53, 323-328 （2004） . 11）Sumner, J. L.; Hopkirk, G. Lipid composition of New Zealand eels. J. Sci. Food Agric. 27, 933-938 （1976）. 12）van Ginneken, V. J. T.; van der Thillart, G. E. E. J. J. Eel fat stores are enough to reach the Sargasso. Nature 403, 156-157 （2000） . 13）Aarestup, K.; Økland, F.; Hansen, M. M.; Righton, D.; Gargan, P.; Castonguay, M.; Bernatchez, L.; Howey, P.; Sparholt, H.; Pedersen, M. I.; McKinley, R. S. Oceanic spawning migration of the European eel （Anguilla an（2009） . guilla） . Science 325, 1660 14）van Ginneken, V. J. T.; Antonissen, E.; Müller, U. K.; Booms, R.; Eding, E., Verreth, J.; van der Thillart, G. E. E. J. J. Eel migration to the Sargasso: remarkably high swimming efficiency and low energy costs. J. Experment. Biol. 208, 1329-1335 （2008）. 15）van Ginneken, V. J. T.; Maes, G. E. The European eel （Anguilla anguilla, Linnaeis） , its lifecycle, evolution and reproduction: a literature review. Rev. Fish. Biol. Fisheries 15, 367-398 （2005） . 16）Chow, S.; Kurogi, H.; Katayama, S.; Anbe, D.; Okazaki, M.; Watanabe, T.; Ichikawa, T.; Kodama, M.; Aoyama, J.; Shinoda, A.; Watanabe, S.; Tsukamoto, K.; Miyazaki, S.; Kimura, S.; Yamada, Y.; Nomura, K.; Tanaka, T.; Kazeto, Y.; Hata, K.; Handa, T.; Tawa, A.; Mochioka, N. Japanese eel Anguilla japonica do not assimilate nutrition during the oceanic spawning migration: evidence from stable isotope analysis. Mar. Ecol. Prog. Ser. 402, 233-238 （2010） . 17）Pankhurst, N. W.; Sorensen, P. W. Degeneration of the alimentary-tract in sexually maturing European Anguilla anguilla（LeSueur）. Can. J. Zool. 62, 11431149 （1984） . 18）Durif, C.; Dufour, S.; Elie, P. The silvering process of Anguilla anguilla: a new classification from the yellow resident to the silver migrating stage. J. Fish. . Biol. 66, 1025-1043 （2005） 19）Ozaki, Y.; Koga, H.; Takahashi, T.; Adachi, S.; Yamauchi, K. Lipid content and fatty acid composition of muscle, liver, ovary and eggs of captive-reared and wild silver Japanese eel Anguilla japonica during artificial maturation. Fisheries Sci. 74, 362-371 （2008）. 20）Oku, T.; Sugawara, A.; Choudhury, M.; Komatsu, M.; Yamada, S.; Ando, S. Lipid and fatty acid compositions differentiate between wild and cultured Japanese eel （Anguilla japonica）. Food Chem. 115, 436-440 （2009） . 21）Folch, J.; Lees, M.; Sloane-Stanley, G. H. A Simple Method for the Isolation and Purification of Total Lip-
ids from Animal Tissues. J. Biol. Chem. 226, 497-509 （1957）. 22）Saito, H.; Sakai, M.; Wakabayashi, T. Characteristics of the lipid and fatty acid compositions of the Humboldt squid, Dosidicus gigas: The trophic relationship between the squid and its prey. Eur. J. Lipid Sci. Technol. 116, 360-366 （2014）. 23）Yu, Q. T.; Liu, B. N.; Zhang, J. Y.; Huang, Z. H. Location of double bonds in fatty acids of fish oil and rat testis lipids. Gas chromatography – mass spectrometry of the oxazoline derivatives. Lipids 24, 79-83（1989）. 24）Shen, K.-N.; Tzeng, W.-N. Population genetic structure of hte year-round spawning tropical eel, Anguilla reinhardtii, in Australia. Zool. Studies 46, 441-453 . （2007） 25）Saito, H.; Hashimoto, J. Characteristics of the fatty acid composition of a deep-sea vent gastropod, Ifremeria nautilei. Lipids 45, 537-548 （2011） . 26）Saito, H.; Murata, M; Hashimoto, J. Lipid characteristics of a seep clam, Mesolinga soliditesta: Comparison with those of two coastal clams, Meretrix lamarckii and Ruditapes philippinarum. Deep-Sea Res. I. 94, 150-158（2014）. 27）Sargent, J. R. Ether-linked glycerides in marine animals, In Ackman, R. G.（ed.）, Marine Biogenic Lipids, Fats, and Oils Vol. I. CRC Press Inc., Florida, pp. 175197（1989）. 28）Nevenzel, J. D.; Gibbs, A.; Benson, A. A. Plasmalogens in the gill lipids of aquatic animals. Comp. Biochem. Physiol. 82B, 293-297（1985）. 29）Deibel, D.; Parrish, C. C.; Grønkjær, P.; Munk, P.; Gissel Nielsen, T. Lipid class and fatty acid content of the leptocephalus larva of tropical eels. Lipids 47, 623634（2012）. 30）Bessonart, M.; Izquierdo, M. S.; Salhi, M.; HernandezCruz, C. M.; Gonzalez, M. M.; Fernandez-Palacios, H. Effectr of dietary arachidonic acid levels on growth and survival of gilthhead sea bream（Sparus aurata L.） larvae. Aquaculture 179, 265-275（1999）. 31）Koven, W.; Barr, Y.; Lutzky, S.; Ben-Atia, I.; Weiss, R. Harel, M., Behrens, P., Tandler, A. The effect of dietary arachidonic acid（20:4n-6）on growth, survival and resistance to handling stress in gilthead seabream（Sparus aurata） larvae. Aquaculture 193, 107-122（2001）. 32）Villalta, M.; Estévez, A.; Bransden, M. P. Arachidonic acid enriched live prey induces albinism in Senegal sole（Solea senegalensis）larvae. Aquaculture 245, 193-209（2005）. 33）Saito, H.; Okabe, M. Characteristic of lipid composition differences between cultured and wild ayu （Plecoglossus altivelis）. Food Chem. 131, 1104-1115（2012）. 34）Dunstan, G. A.; Baillie, H. J.; Barrett, S. M.; Volkman, J.K. Effect of diet on the lipid composition of wild and cultured abalone. Aquaculture 40, 115-127（1996）. 615
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35）Saito, H.; Aono, H. Characteristic of lipid and fatty acid of marine gastropod Turbo cornutus: High levels of arachidonic and n-3 docosapentaenoic acid. Food Chem. 145, 135-144 （2014） . 36）Owen, J. M.; Adron, J. W.; Middleton, C.; Cowey, C. B. Elongation and desaturation of dietary fatty acids in turbot Scophthalmus maximus L., and rainbow trout Salmo gairdnerii Rich. Lipids 10, 528-531 （1975）.
37）Yamada, K.; Kobayashi, K.; Yone, Y. Conversion of linolenic acid to ω3-highly unsaturated fatty acids in marine fishes and rainbow trout. Bull. Jap. Soc. Sci. Fish. 46, 1231-1233（1980）. 38）Tocher, D. R. Fatty acid requirements in ontogeny of marine and freshwater fish. Aquacul. Res. 41, 717732（2010）.
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Variation of Lipids and Fatty Acids of the Japanese Freshwater Eel, Anguilla japonica, during Spawning Migration Hiroaki Saito1＊ , Hiroaki Kurogi2, Seinen Chow3 and Noritaka Mochioka4 1
Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, 921-8836, Japan. Yokosuka-Chosha, National Research Institute of Aquaculture, 6-3-1 Nagai, Yokosuka 238-0316, Japan. 3 National Research Institute of Fisheries Science, 2-12-4, Fukuura, Kanazawa-Ku, Yokohama 236-8648, Japan. 4 Kyushu University, 6-10-1, Hakozaki, Fukuoka 812-8581, Japan. 2
Abstract: The lipid and fatty acid composition of the muscle of the wild Japanese freshwater eel, Anguilla japonica, was analyzed between the initial and terminal stages of spawning migration to clarify the relationship between lipid physiology and maturation. Triacylglycerols were the only major component in the initial-phase eels, which contained high levels of lipids, while comparatively low triacylglycerol levels were observed in terminal-phase eels (Mariana silvers) at spawning area. Significant levels of plasmalogens were found in its phosphatidylethanolamine, different from other common fish species, which have their little levels. The major fatty acids in A. japonica depot triacylglycerols were 14:0, 16:0, 18:0, 16:1n-7, 18:1n7, 18:1n-9, and 18:2n-6. Noticeable levels of 20:4n-6, EPA, 22:5n-3, and DHA were also found in initialphase sample TAG at the yellow and initial silver stages. High 18:2n-6 levels in all A. japonica lipids were similar to those in other common freshwater fishes. In all A. japonica tissue phospholipids, high levels of n-6 and n-3 PUFA, such as 20:4n-6, EPA, 22:5n-3, and DHA, were observed except for the matured terminal female sample. High n-6 PUFA levels in terminal-phase samples caught at the spawning area suggest that A. japonica maintains and uses initial fatty acids from inland waters without feeding during long spawning migrations. The post-spawning sample, containing low levels of 20:4n-6 and DHA with unusually high levels of its degradation products (18:3n-6, 20:2n-6, and 18:4n-3), indicates that A. japonica may finally use its most important PUFA as energy for spawning before ending its life. Key words: arachidonic acid, chemoecology, spawning migration, fatty acid, marine lipid, monounsaturated fatty acid, polyunsaturated fatty acid 1 INTRODUCTION Low levels of n-6 PUFA are generally found in marine fish lipids. For example, 18:2n-6 and 20:4n-6 are minor components in their lipids1−5）, except for some marine herbivorous animals that prefer and consume seaweed and specifically accumulate 20:4n-6 in their tissue lipids6, 7）. In contrast, freshwater fishes consume terrestrial prey. Therefore, their lipids specifically contain high levels of n-6 PUFA. The n-6 PUFA are always observed as major PUFA in terrestrial animal lipids. For example, 18:2n-6 and 20:4n-6 are characteristically contained in freshwater fishes. In particular, 18:2n-6 is observed in their depot neutral lipids and 20:4n-6 is mainly found in the tissue phospholipids of fish such as common carp, Cyprinus
carpio8, 9）, Carassius auratus auratus9）; rainbow trout, Salmo gairdnerii8）; eel, Anguilla japonica8）; and Indian featherback fish, Notopterus notopterus10）. This suggests that 18:2n-6 and 20:4n-6 are useful as lipid biomarkers for terrestrial8−10）or herbivorous animals6, 7）. Wild eels（Anguilla spp., Anguillidae）mainly dwell in Abbreviations: ARA, arachidonic acid; DMOX, 4,4-dimethyloxazoline; DHA, docosahexaenoic acid; EPA (IPA); icosapentaenoic acid; GC/MS, gas chromatography/mass spectroscopy; MUFA, monounsaturated fatty acids; NMID, non-methylene-interrupted dienes; NMR, nuclear magnetic resonance; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PUFA, polyunsaturated fatty acids; TAG, triacylglycerols; TFA, total fatty acids; TL, total lipids.
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Correspondence to: Hiroaki Saito, Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, 921-8836, Japan. E-mail: [email protected] Accepted February 2, 2015 (received for review December 26, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 603
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freshwater areas, take prey in lakes and rivers, and their fatty acids contain significant levels of 18:2n-6 and 20:4n68, 11）. Freshwater eels of the genus Anguilla metamorphose into the silver stage from yellow stage upon onset of maturation, cease feeding and start reproductive migration. European eels（Anguilla anguilla）undertake a 5000-km spawning migration from Europe to the Sargasso Sea12, 13）, and details of their migration have been gradually revealed. Laboratory experiments support the fact that European eels are capable of swimming a distance of over 5500 km without resting and feeding14） and the starving silver eels are able to reach their spawning grounds by using their stores of internal fat15）. In the Pacific Ocean, the Japanese eel （Anguilla japonica Temminck & Schlegel, Anguillidae） may also depend on its store of fat for spawning migrations without feeding16） because the alimentary tracts of silver eels are degenerated17, 18）. Recently, adult matured Japanese eels（terminal phase）have been caught in the West Mariana Ridge, which is its spawning area16）. An interesting comparison of the fatty acids of spawning-area eels（terminal phase） can be made with those of Japanese eels at the , which are collected yellow and silver stages （initial phase） in rivers and lakes and presumably influenced by terrestrial . prey lipids（Lake Ogawara19）, and Amori River20）） The present manuscript deals with the detailed lipid composition and analysis of the fatty acid components of the major lipid classes of A. japonica between these ‘Mariana silvers’ （terminal phase） and Japanese eels at the yellow and initial silver stages（initial phase）in order to clarify its lipid physiology and to find out its feeding behavior during reproductive migrations.
2 MATERIALS AND METHODS 2.1 Materials Specimens of yellow and initial silver stages A. japonica （yellow-stage eels: samples No. 1–4, silver-stage eels: samples No. 5–10 in Table 1）were caught in three Japanese rivers（the Koyama River, the Yakkan River, and the Akigawa River）and Okinoshima Island near Japanese coasts of the Japan Sea and the Beppu Bay in Seto-Inland Sea in the northern Pacific Ocean. Specimens of completely matured Mariana silver eel A. japonica（terminal-phase stage eels: samples No. 11–17 in Table 1）were caught by the research vessels‘Kaiyo-maru’and“Hokko-maru”, which belongs to the Fisheries Agency, Tokyo, Japan, in the West Mariana Ridge in the mesopelagic zone of the northwestern Pacific Ocean. Research cruises were performed during 2008 and 2009, and adult Japanese eels were captured by a large mid-water trawl net in the southern part of the West Mariana Ridge（ Table 1）16）. The samples of A. japonica listed in Table 1 were frozen at −40℃ until extraction. After measurements of body
length and weight of A. japonica samples, the following tissues and organs, such as muscle and liver, were dissected from each fish. 2.2 Lipid extraction and analysis of lipid classes Each individual（muscle）was immersed in a reagent mixture containing chloroform and methanol（2:1, v/v）, and homogenized in the same solvents, and each lipid of a homogenized sample was then extracted according to the Folch procedure21）. Each crude lipid was separated into classes on a silicic acid column（Merck and Co. Ltd., Kieselgel 60, 70 - 230 mesh）, and the constituent lipids were quantitatively analyzed by gravimetric analysis of the column chromatography fractions（Table 2）22）. The lipid classes from each lipid fraction were identified by comparison of the retention factor（Rf）values of standards using plate thin-layer chromatography （Merck & Co. Ltd., Kieselgel 60, thickness of 0.25 mm for analysis） , and by the characteristic peaks observed in nuclear magnetic resonance 22） . All sample lipids were dried under argon at room （NMR） temperature, and stored at −40℃ under an argon atmosphere. 2.3 NMR spectrometry and the determination of lipid classes Spectra were recorded on a GSX-270 NMR spectrometer （JEOL Co. Ltd., Tokyo, Japan）in a pulsed Fourier transform mode at 270 MHz in a deuterochloroform solution using tetramethylsilane as the internal standard22）. 2.4 Preparation of methyl esters and analysis by gasliquid chromatography Fatty acids present in TAG, PE, and PC fractions were converted to fatty acid methyl esters by direct transesterification with methanol containing 1％ concentrated hydrochloric acid under reflux for 1.5 hr, as previously reported22）. These methyl esters were purified using silica gel column chromatography by elution with dichloromethane/ n-hexane（ 2/1, v/v）. The composition of the fatty acid methyl esters were determined by gas liquid chromatography using an HP 6890N gas chromatograph（Agilent Technology Co., Yokogawa Electric Corporation, Tokyo, Japan） equipped with an omegawax-250 fused silica capillary column（30 m×0.25 mm i. d. ; 0.25 μm film, Supelco Japan Co. Ltd, Tokyo, Japan）. The temperatures of the injector and the column were held at 250 and 210℃, respectively, and the split ratio was 1:76. Helium was used as the carrier gas at a constant inlet rate of 0.7 mL/min. Quantitation of individual components was performed by means of an HP ChemStation System （Rev. B.03.02 ［341］version, Yokogawa HP Co. Ltd.） electronic integrator（Tables 3–6）.
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Table 1 Locality of capture and biological data of three types of wild Japanese eel, Anguilla japonicus.
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Table 2 The lipid contents and lipid classes of the muscles of three types of wild Japanese eel, Anguilla japonica.
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Table 3 The fatty acid composition of muscles of the common yellow Eels (%)a.
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Table 4 The fatty acid composition of muscles of the silver Eels (%)a.
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Table 5 The fatty acid composition of muscles of the mature 2008 eel (%)a.
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Table 6 The fatty acid composition of muscles of the mature 2009 eel (%)a.
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2.5 Preparation of 4,4-dimethyloxazoline derivatives （DMOX）and analysis of DMOX by gas chromatography - mass spectrometry （GC-MS） DMOX derivatives were prepared by adding an excess amount of 2-amino-2-methylpropanol to a small amount of the fatty acid methyl esters in a test tube under an argon atmosphere. The mixture was heated at 180℃ for 18h22, 23）. Analysis of the DMOX derivatives was performed on an HP G1800C GCD Series II GC-MS（Hewlett Packard Co. Ltd., Yokogawa Electric Corporation, Tokyo, Japan）and an Agilent 6890N-5973N GC-MS System（Agilent Technologies Inc., Tokyo, Japan）equipped with the same capillary column for determining the fatty acids with an HP WS（HP Kayak XA, G1701BA version, PC workstations and MSD ChemStation D.03.00.611） . The temperatures of the injector and the column were held at 230 and 210℃, respectively. The split ratio was 1:76 and the ionization voltage was 70 eV. Helium was used as the carrier gas at a constant inlet rate of 0.7 mL/min. Fatty acid methyl esters were identified by comparing the mass spectral data of the methyl esters and DMOX derivatives obtained by GC-MS. 2.6 Statistical analyses The determination of fatty acids by gas-liquid chromatography was based on more than two replicates（n＝2–12 in Tables 3–6）. Lipid content, lipid class, and fatty acid levels（％ of wet weight, ％ of total lipids, and ％ of total fatty acids） are expressed as mean values±standard errors. The mean total PUFA, total n-4 PUFA, total n-6 PUFA, total n-3 PUFA levels of the samples were compared with each other. Significant mean differences were determined using a one-way analysis of variance（ANOVA）. Tukey’ s multiple procedure was used to compare the differences among mean values. Differences were regarded as significance level of p＜0.05.
3 RESULTS 3.1 Lipid content in the three different types of A. japonica The three types of A. japonica samples（yellow stage samples 1–4: silver stage samples 5–10: and Mariana silver samples 11–17）were caught in the Japan Sea and the northwestern Pacific Ocean, as shown in Table 1. Except for sample 4, the muscle lipid content in two types of samples caught in rivers and near Japanese coasts was significantly high（ samples 1–3: 12.8–16.3％ and samples 5–10: 14.4–20.6％）, while that in matured Mariana silver samples was comparatively low and fluctuated（ 2008 samples 11–14: 0.3–2.3％ and 2009 samples 15–17: 3.2– 9.7％ in Table 2）.
lipids（steryl esters, wax esters, diacylglyceryl ethers, TAG, sterols, and free fatty acids）and phospholipids（PE and PC）, as shown in Table 2. In all muscles of A. japonica caught near Japan（initial phase）, TAG（69.7– 94.0％ for samples 1–4 and 93.2–95.5％ for samples 5–10）was the only major component with trace or small levels of phospholipids. Medium levels of free fatty acids were found in two terminal-phase samples（13.4％ for sample 11 and 18.3％ for sample 13. Unusually medium levels of dimethyl acetals （DMA） in all muscle PE（13.4–22.0％ for samples 1–4, 9.5– 17.4％ for samples 5–10, and 3.1–15.2％ for samples 11–17） were observed（Tables 3–6） . 3.3 Fatty acid composition of A. japonica major TAG The mean values of the fatty acid compositions of the major purified lipids extracted from A. japonica are presented in Tables 3–7. At least fifty peaks were detected and identified, of which more than thirty consisted of more than 0.1％ total fatty acids（TFA）. Even-numbered carbon atoms, ranging from C14 to C22, were more abundant than odd-numbered and branched ones, which is similar to those of all other fish species. The kinds of fatty acids of all phases of A. japonica TAG were similar to each other. In the final-phase samples caught near the West Mariana Ridge, seven major components（more than about 3％ of TFA）were found in the final-phase A. japonica depot TAG, 14:0, 16:0, 18:0, 16:1n-7, 18:1n-7, 18:1n-9, and 18:2n-6, which are mostly saturated and monounsaturated fatty acids. Although the TAG contained various kinds of fatty acids, these seven components were detected at a level of about 3％ or more of TFA of the final-phase specimens. Noticeable levels of n-6 and n-3 long-chain PUFA such as 20:4n-6, EPA, 22:5n-3, and DHA were also found in the TAG of some final-phase samples. In contrast, several major PUFA were found in the TAG of the fatty initialphase samples, which were caught in the rivers and near Japanese coasts: EPA and 22:5n-3 with noticeable levels of some other PUFA such as 18:2n-6, 20:4n-6, 18:3n-3, and DHA. The levels of these PUFA were sometimes over about 3％ of TFA, in particular, in the case of EPA and 22:5n-3 in these samples（samples 1–3, 5, and 10） . 3.4 Fatty acid composition of A. japonica tissue phospholipids In the phospholipids of all samples, nine major components were found uniformly, 16:0, 18:0, 16:1n-7, 18:1n-7, 18:1n-9, 20:4n-6, EPA, 22:5n-3, and DHA, with noticeable levels of 14:0, 18:2n-6, and 18:3n-6, except for the terminal sample, No. 13, whose major fatty acids were expanded to various short-chain fatty acids, 14:0, 16:0, 18:0, 18:1n-9, 18:2n-6, 18:3n-6, 20:2n-6, 20:4n-6, 18:4n-3, and DHA.
3.2 Lipid Classes of A. japonica The TL were fractionated into various classes as neutral 611
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Table 7 Actual DHA levels in 100 g of wet tissue.
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Variation of Lipids and Fatty Acids of the Japanese Freshwater Eel, Anguilla japonica, during Spawning Migration
4 DISCUSSION 4.1 Lipid content in three different types of A. japonica A. japonica caught near Japan （initial phase） accumulated high levels of lipids in the muscles: this characteristic feature is also generally observed in the other Anguilla species10, 19）. However, after long-distance spawning migrations, the Mariana silver eels（terminal phase）have lower lipid levels（ Table 2）. For example, samples 11 and 12 （male, 2008） and samples 13 and 14 （female, 2008） , having the lowest lipid levels completely lost their lipids, while samples 15 and 16（male, 2009）and sample 17（female, 2009）, which have several percentages of lipid content, may be able to maintain their lives. This difference in the Mariana silvers between 2008 and 2009 indicates that A. japonica may partly use its internal fat stores for long-distance migration over 2000–3000 km12, 14, 16） and may retain its depot lipids for multiple spawnings 24）. The 2008 samples, which completely lost their lipids（0.3–2.3％ for 11–14）may be exhausted by migration and spawning, while the 2009 samples（ 15–17）may be able to spawn several times and be close to ending its life. 4.2 Lipid Classes of A. japonica The occurrence of high levels of muscle TAG was due to the high levels of muscle lipid content because phospholipid levels in animal cells remain constant and the relative levels of depot lipids in fatty fishes are generally higher than those in lean animals4−7, 25, 26）. Medium levels of free fatty acids were specifically found in two terminal-phase, which probably degenerated from TAG to decomposed free fatty acids. The enzymatic degradation of A. japonica TAG may have actively proceeded at the terminal phase because a terminal-phase eel, which is probably unable to feed17, 18）, should obtain energy for spawning from its internal tissue lipids. Different from other fish species27, 28）, unusually medium levels of dimethyl acetals（ DMA）in muscle PE suggest that plasmalogen-type PE are biosynthesized in eel muscles similar to those in invertebrates such as mollusks25, 26）. A. japonica mainly biosynthesizes DMA 16:0, 18:0, and 18:1 for alkenyl ethers in PE plasmalogens, which is also similar to those of other common invertebrates25−27）. This is the characteristic profile of the freshwater Japanese eel. 4.3 Fatty acid composition of A. japonica major TAG High 18:2n-6 levels in all A. japonica TAG lipids were similar to those in other common freshwater fishes8−10）. The characteristically high levels of 18:2n-6 in their TAG are presumably influenced by those of the prey lipids in inland waters. Similar to the lipids of freshwater eel, carp8, 9）, and featherback 10）, which are typical freshwater fish species in rivers and lakes, all A. japonica had high levels of 18:2n-6. For example, its levels in the TAG of A. japonica of both phases were 1.5–5.3％ for samples 1–4, 1.0– 6.6％ for samples 5–10, 2.7–3.9％ for samples 11–14, and
2.0–3.1％ for samples 15–17. In particular, samples 11–17 showed comparatively high 18:2n-6 levels in the TAG even though they have inhabited marine environments for a long time1−4）. Simultaneously, the terminal-phase samples have markedly low levels of long-chain n-3 PUFA（EPA: 0.1– 1.2％ for samples 11–14 and 0.2–1.3％ for samples 15–17; DHA: 0.4–1.4％ for samples 11–14 and 0.7–2.0％ for samples 15–17）, different from those in other common marine fish species, which feed on marine prey, assimilate their lipids, and have high levels of these n-3 PUFA1−5, 29）. For example, high levels of DHA are usually observed in typical marine fishes, such as tuna, which are highly migratory fishes1, 2, 4）. These findings imply that the terminalphase samples are probably not dependent on marine prey lipids and are continuously influenced by only inland animal lipids15）. 4.4 Fatty acid composition of A. japonica tissue phospholipids Although the lipid contents between initial-phase samples（1–10）and terminal-phase samples（11–17）were different from each other, various n-6 PUFA（18:2 n-6, 18:3 n-6, 20:2 n-6, 20:3 n-6, and 20:4 n-6）and high levels of long-chain n-3 PUFA（EPA, 22:5 n-3 and DHA）in all sample PE were observed in both phases of the samples except for the terminal sample, No. 13. Similarly, the same trends were also found in PC, except for the terminal sample, No. 13. In particular, consistently high levels of 20:4n-6 and DHA in all sample PE and PC were observed in both phases of the samples. The 20:4n-6 levels in the phospholipids of A. japonica samples ranged as follows: 4.6–14.2％ for PE and PC of samples 1–4, 4.6–12.3％ for those of samples 5–10, 7.2–16.8％ for those of samples 11, 12, and 14, and 5.1–11.3％ for those of samples 15–17. The same trends are found for DHA: 8.0–19.1％ for PE and PC of samples 1–4, 9.5–20.4 for those of samples 5–10, 9.4–22.8％ for those of samples 11, 12, and 14, and 15.4–28.7％ for those of samples 15–17 except for the terminal sample, No. 13, whose n-3 and n-6 LC PUFA levels were the lowest. These phenomena suggest the importance and essentiality of 20:4n-6 and DHA for A. japonica 29−32） although even 20:4n-6 and DHA were consumed in the case of sample No. 13, which was presumably in its last mortal stage. As for LC n-3 PUFA, the same trends were observed in the marine eels, which have high levels of both EPA and DHA29）. 4.5 High levels of n-6 PUFA and degradation of 20:4n-6 and DHA in A. japonica tissue phospholipids Similar to other freshwater fishes9, 10）, the significant level of 20:4n-6 of all A. japonica polar lipids differed from that of common marine fish species1−5）. Different from the fatty acid compositions of marine origin larvae of tropical eels29）, high 20:4n-6 levels in the major polar lipids indicate 613
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that A. japonica is not dependent on marine prey animal lipids, which contain low levels of n-6 PUFA and high levels of n-3 PUFA1−5） except for the terminal sample, No. 13. With significant 18:2n-6 levels in TAG, the high levels of 20:4n-6 in polar lipids in terminal-phase A. japonica also indicate that A. japonica shows profiles of typical freshwater animal lipids even in the sea. This finding supports the assumption that A. japonica, which is carnivorous, migrates to spawning areas without feeding16） because high n-6 PUFA levels are only found in some marine herbivorous fishes6, 7）. In general, limited long-chain n-3 PUFA, such as EPA and DHA, are mainly found in marine fishes in higher trophic levels. In particular, DHA is the only major PUFA in highly migratory fishes1, 2, 4）. This means that both the EPA and DHA are terminal PUFA and many marine fishes only accumulate them1−5）. Interestingly, unusually high levels of 22:5n-3 were found in the polar lipids of all A. japonica. This is the specific lipid profile for A. japonica because only some mollusks, such as abalone and snails25, 34, 35）, show noticeable levels of 22:5n-3. High levels of 22:5n-3 in A. japonica lipids indicate its biosynthetic weakness of DHA, similar to those in the mollusks25, 34, 35） and other freshwater fishes8, 33）. In the spawning migration, the actual DHA levels in the tissue of A. japonica were markedly reduced in terminalphase eels （2008 Mariana silver eels 11–14） , which had finished migration and spawning, while those of A. japonica after migration（ 2009 Mariana silver eels 15–17）were similar to those in initial-phase eels. For example, the actual DHA amounts in the depot TAG were 0.4–1.4 mg （lipid content×TAG ratio×DHA ratio/100 g of wet tissue） in the final phase（the 2008 eels 11–14）while those were 34.6–369.7 mg and 160.8–644.9 mg in the initial phase （yellow eels 1–4 and silver eels 5–10） . In the case of 2009 samples （15–17） , the actual DHA amounts were 15.9–171.2 mg, similar to those in the two initial phase samples （Table 7） . Similar trends were observed in the phospholipids. For example, the actual DHA amounts in the tissue PE （lipid content×PE ratio×DHA ratio/100 g of wet tissue）at the final phase samples （the 2008 eels 11–14） were 3.6–5.8 mg, while those at the two initial phases（ samples 1–4 and 5–10）were 10.9–46.1, 3.8–17.8, and at the final phase samples（the 2009 eels 15–17）were 14.0–43.7 mg（Table 7）. This implies that A. japonica probably retain DHA during spawning migration by using other fatty acids such as saturated fatty acids and MUFA as energy sources. Eventually, however, it finally uses even the most important PUFA, DHA36−38）, for multiple spawnings, and then ends its life.
ACKNOWLEDGMENTS We are especially indebted to the captains and crew of the Research Vessels“Kaiyo-maru”and“Hokko-maru” of the Fisheries Agency for their cooperation and enthusiasm throughout the work. The authors also thank Ms. Sumiko Terada, Ms. Noriko Tsutsui, and Mr. Akihito Takashima for their skilled technical assistance. This work was supported in part by the research projects from the Fisheries Research Agency of Japan. H. S. performed all aspects of the study including the design of the experiments, analyzing the data, and writing the manuscript. H. K., S. C., and N. M. caught the animals and determined biological data.
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